Methods and compositions for producing secreted trimeric receptor analogs and biologically active fusion proteins

ABSTRACT

Methods and compositions for producing secreted soluble receptors and biologically active polypeptides in trimeric forms are disclosed. The process involves fusing the DNA template encoding a soluble receptor with a ligand binding domain or biologically active polypeptide to a DNA sequence encoding a C-propeptide of collagen, which is capable of self-assembly into a covalently linked trimer. The resulting fusion proteins are secreted as trimeric soluble receptor analogs, which can be used for more efficient neutralization of the biological activities of their naturally occurring trimeric ligands.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.10/677,877 filed on Oct. 2, 2003, now U.S. Pat. No. 7,268,116.

FIELD OF THE INVENTION

The present invention relates to methods for protein expression, andmore specifically, for creating and expressing secreted and biologicallyactive trimeric proteins, such as trimeric soluble receptors.

BACKGROUND OF INVENTION

In multicellular organisms, such as humans, cells communicate with eachother by the so-called signal transduction pathway, in which a secretedligand (e.g. cytokines, growth factors or hormones) binds to its cellsurface receptor(s), leading to receptor activation. The receptors aremembrane proteins, which consist of an extracellular domain responsiblefor ligand binding, a central transmembrane region followed by acytoplasmic domain responsible for sending the signal downstream. Signaltransduction can take place in the following three ways: paracrine(communication between neighboring cells), autocrine (cell communicationto itself) and endocrine (communication between distant cells throughcirculation), depending on the source of a secreted signal and thelocation of target cell expressing a receptor(s). One of the generalmechanisms underlying receptor activation, which sets off a cascade ofevents beneath the cell membrane including the activation of geneexpression, is that a polypeptide ligand such as a cytokine, is presentin an oligomeric form, such as a homo- dimer or trimer, which when boundto its monomeric receptor at the cell outer surface, leads to theoligomerization of the receptor. Signal transduction pathways play a keyrole in normal cell development and differentiation, as well as inresponse to external insults such as bacterial and viral infections.Abnormalities in such signal transduction pathways, in the form ofeither underactivation (e.g. lack of ligand) or overactivation (e.g. toomuch ligand), are the underlying causes for pathological conditions anddiseases such as arthritis, cancer, AIDS, and diabetes.

One of the current strategies for treating these debilitating diseasesinvolves the use of receptor decoys, such as soluble receptorsconsisting of only the extracellular ligand-binding domain, to intercepta ligand and thus overcome the overactivation of a receptor. The bestexample of this strategy is the creation of Enbrel®, a dimeric solubleTNF-α receptor-immunoglobulin (IgG) fusion protein by Immunex (Mohler etal., 1993; Jacobs et al., 1997), which is now part of Amgen. The TNFfamily of cytokines is one of the major pro-inflammatory signalsproduced by the body in response to infection or tissue injury. However,abnormal production of these cytokines, for example, in the absence ofinfection or tissue injury, has been shown to be one of the underlyingcauses for diseases such as arthritis and psoriasis. Naturally, a TNF-αreceptor is present in monomeric form on the cell surface before bindingto its ligand, TNF-α, which exists, in contrast, as a homotrimer(Locksley et al., 2001). Accordingly, fusing a soluble TNF-α receptorwith the Fc region of immunoglobulin G1, which is capable of spontaneousdimerization via disulfide bonds (Sledziewski et al., 1992 and 1998),allowed the secretion of a dimeric soluble TNF-α receptor (Mohler etal., 1993; Jacobs et al., 1997). In comparison with the monomericsoluble receptor, the dimeric TNF-α receptor II-Fc fusion has a greatlyincreased affinity to the homo-trimeric ligand. This provides amolecular basis for its clinical use in treating rheumatoid arthritis(RA), an autoimmune disease in which constitutively elevated TNF-α, amajor pro-inflammatory cytokine, plays an important causal role.Although Enbrel® was shown to have a Ki in the pM range (μg/mL) to TNF-α( Mohler et al., 1993), 25 mg twice a week subcutaneous injections,which translates to μg/mL level of the soluble receptor, are requiredfor the RA patients to achieve clinical benefits (www.enbrel.com). Thehigh level of recurrent Enbrel® consumption per RA patients has createda great pressure as well as high cost for the drug supply, which limitsthe accessibility of the drug to millions of potential patients in thiscountry alone.

In addition to the TNF-α family of potent proinflammatory cytokines, theHIV virus that causes AIDS also uses a homo-trimeric coat protein,gp120, to gain entry into CD-4 positive T helper cells in our body(Kwong et al., 1998). One of the earliest events during HIV infectioninvolves the binding of gp120 to its receptor CD-4, uniquely expressedon the cell surface of T helper cells (Clapham et al., 2001). Monomericsoluble CD-4 was shown over a decade ago as a potent agent against HIVinfection (Clapham et al., 1989) however, the excitement was sadlydashed when its potency was shown to be limited only to laboratory HIVisolates (Daar et al., 1990). It turned out that HIV strains from AIDSpatients, unlike the laboratory isolates, had a much lower affinity tothe monomeric soluble CD-4, likely due to the sequence variation on thegp120 (Daar et al., 1990). Although the dimeric soluble CD-4-Fc fusionproteins have been made, these decoy CD-4 HIV receptors showed littleantiviral effect against natural occurring HIVs from AIDS patients, bothin the laboratories and in clinics, due to the low affinity to the gp120(Daar et al., 1990).

Clearly, there is a great need to be able to create secretedhomo-trimeric soluble receptors or biologically active proteins, whichcan have perfectly docked binding sites, hence higher affinity, to theirnaturally occurring homo-trimeric ligands, such as the TNF family ofcytokines and HIV coat proteins. Such trimeric receptor decoystheoretically should have a much higher affinity than its dimericcounterparts to their trimeric ligand. Such rationally designed solubletrimeric receptor analogs could significantly increase the clinicalbenefits as well as lower the amount or frequency of the drug injectionsfor each patient. To be therapeutically feasible, a desired trimerizingprotein moiety for biologic drug designs should satisfy the followingcriteria. Ideally it should be part of a naturally secreted protein,like immunoglobulin Fc, that is also abundant (non-toxic) in thecirculation, human in origin (lack of immunogenicity), relatively stable(long half-life), capable of efficient self-trimerization which isstrengthened by inter-chain covalent disulfide bonds, and pertain anoptimal geometry in projecting soluble receptor to be trimerized toconfirm maximum ligand binding.

Collagen is a family of fibrous proteins that are the major componentsof the extracellular matrix. It is the most abundant protein in mammals,constituting nearly 25% of the total protein in the body. Collagen playsa major structural role in the formation of bone, tendon, skin, cornea,cartilage, blood vessels, and teeth (Stryer, 1988). The fibrillar typesof collagen I, II, III, IV, V, and XI are all synthesized as largertrimeric precursors, called procollagens, in which the centraluninterrupted triple-helical domain consisting of hundreds of “G-X-Y”repeats (or glycine repeats) is flanked by non-collagenous domains (NC),the N- propeptide and the C-propeptide (Stryer, 1988). Both the C- andN-terminal extensions are processed proteolytically upon secretion ofthe procollagen, an event that triggers the assembly of the matureprotein into collagen fibrils which forms an insoluble cell matrix(Prockop et al., 1998). BMP-1 is a protease that recognizes a specificpeptide sequence of procollagen near the junction between the glycinerepeats and the C-prodomain of collagens and is responsible for theremoval of the propeptide (Li et al.). The shed trimeric C-propeptide oftype I collagen is found in human sera of normal adults at aconcentration in the range of 50-300 ng/mL, with children having a muchhigher level which is indicative of active bone formation (Melkko etal.). In people with familial high serum concentration of C-propeptideof type I collagen, the level could reach as high as 1-6 μg/mL with noapparent abnormality, suggesting the C-propeptide is not toxic (Sorva etal.). Structural study of the trimeric C-propeptide of collagensuggested that it is a tri-lobed structure with all three subunitscoming together in a junction region near their N-termini to connect tothe rest of the procollagen molecule (Bernocco et al.). Such geometry inprojecting proteins to be fused in one direction is similar to that ofFc dimer.

Type I, IV, V and XI collagens are mainly assembled into heterotrimericforms consisting of either two α-1chains and one α-2 chain (for Type I,IV, V), or three different a chains (for Type XI), which are highlyhomologous in sequence. The type II and III collagens are bothhomotrimers of α-1chain. For type I collagen, the most abundant form ofcollagen, stable α-1(I) homotrimer is also formed and is present atvariable levels (Alvares et al., 1999) in different tissues. Most ofthese collagen C-propeptide chains can self-assemble into homotrimers,when over-expressed alone in a cell. Although the N-propeptide domainsare synthesized first, molecular assembly into trimeric collagen beginswith the in-register association of the C-propeptides. It is believedthe C-propeptide complex is stabilized by the formation of interchaindisulfide bonds, but the necessity of disulfide bond formation forproper chain registration is not clear. The triple helix of the glycinerepeats and is then propagated from the associated C-termini to theN-termini in a zipper-like manner. This knowledge has led to thecreation of non-natural types of collagen matrix by swapping theC-propeptides of different collagen chains using recombinant DNAtechnology (Bulleid et al., 2001). Non-collagenous proteins, such ascytokines and growth factors, also have been fused to the N-termini ofeither pro-collagens or mature collagens to allow new collagen matrixformation, which is intended to allow slow release of the noncollagenousproteins from the cell matrix (Tomita et al., 2001). However, under bothcircumstances, the C-propeptides are required to be cleaved beforerecombinant collagen fibril assembly into an insoluble cell matrix.

Although, other protein trimerization domains, such as those from GCN4from yeast (Yang, X. et al, 2000), fibritin from bacteria phage T4(Frank, S. et al., 2001) and aspartate transcarbamoylase of Escherichiacoli (Chen, B. et al., 2004), have been described previously to allowtrimerization of heterologous proteins, none of these trimerizingproteins are human in nature, nor are they naturally secreted proteins.As such, any trimeric fusion proteins would have to be madeintracellularly, which not only may fold incorrectly for naturallysecreted proteins such as soluble receptors, but also make purificationof such fusion proteins from thousands of other intracellular proteinsdifficult. Moreover, the fatal drawback of using such non-human proteintrimerization domains (e.g. from yeast, bacteria phage and bacteria) fortrimeric biologic drug design will be their immunogenicity in the humanbody, rendering such fusion proteins ineffective within weeks afterinjecting into the human body.

One secreted protein previously used as a protein trimerization tag istetranectin, which is a plasminogen-binding protein of C-lectin family(Holtet et al.). However, unlike IgG Fc dimerization tag, the trimerictetranectin structure is not strengthened by any interchain disulfidebonds, and significant fractions of both monomeric and dimerictetranectin co-existed with the trimeric structure in solution (Holtetet al.). Physiologically, teranectin is involved in tissue remodelingand increased cell matrix concentration of tetranectin in human has beenlinked to multiple cancer types. Recombinant heterologous tetranectinfusion proteins have only been produced intracellularly in E. coli asinsoluble inclusion bodies that required refolding to obtain solublestructures (Holtet et al. and Graversen et al.). These unfavorableattributes suggest that tetranectin is not ideal for therapeuticapplications as a protein trimerization tag. Nonetheless, bacteriallyproduced ApoAI-Tetranectin fusion protein has been produced and patented(Graversen et al.) and is being tested as a therapeutic agent foratherosclerosis.

SUMMARY OF THE INVENTION

Disclosed here is an invention that allows any soluble receptors orbiologically active polypeptides to be made into trimeric forms assecreted proteins. The essence of the invention is to fuse any solublereceptors and biologically active proteins in-frame to the C-propeptidedomain of fibrillar collagen, which is capable of self-trimerization,using recombinant DNA technology. The resulting fusion proteins whenexpressed in eukaryotic cells are secreted as soluble proteinsessentially 100% in trimeric forms covalently strengthened byinter-molecular disulfide bonds formed among three C-propeptides.

In one aspect of the invention, a method for producing secreted trimericfusion proteins is disclosed, comprising the following: (a) introducinginto a eukaryotic host cell a DNA construct comprising a promoter whichdrives the transcription of an open reading frame consisting of a signalpeptide sequence which is linked in-frame to a non-collagen polypeptideto be trimerized, which in turn is joined in-frame to the C-terminalportion of collagen capable of self-trimerization; (b) growing the hostcell in an appropriate growth medium under physiological conditions toallow the secretion of a trimerized fusion protein encoded by said DNAsequence; and (c) isolating the secreted trimeric fusion protein from ahost cell.

Within one embodiment, the signal peptide sequence is the nativesequence of the protein to be trimerized. Within another embodiment, thesignal peptide sequence is from a secreted protein different from thatto be trimerized. Within one embodiment, the non-collagen polypeptide tobe trimerized is a soluble receptor consisting of the ligand bindingdomain(s). Within one embodiment, the C-terminal portion of collagen isthe C-propeptide without any triple helical region of collagen (SEQ IDNOS: 3-4 and SEQ ID NOS: 17-18). Within another embodiment, theC-terminal collagen consists of a portion of the triple helical regionof collagen as linker to the non-collagenous proteins to be trimerized(SEQ ID NOS. 1-2). Within another embodiment, the C-terminal portion ofcollagen has a mutated or deleted BMP-1 protease recognition site (SEQID NOS. 3-4 and SEQ ID NOS: 17-18).

In one aspect of the invention, a method for producing a secretedtrimeric fusion protein is disclosed, comprising the following: (a)introducing into a eukaryotic host cell a DNA construct comprising apromoter which drives the transcription of an open reading frameconsisting of a signal peptide sequence which is linked in-frame to anon-collagen polypeptide to be trimerized, which in turn is joinedin-frame to the C-terminal portion of collagen capable ofself-trimerization, selected from pro.alpha.1(I), pro.alpha.2(I),pro.alpha.1(II), pro.alpha.1(III), pro.alpha.1(V), pro.alpha.2(V),pro.alpha.1(XI), pro.alpha.2(XI) and pro.alpha.3(XI); (b) growing thehost cell in an appropriate growth medium under physiological conditionsto allow the secretion of a trimerized fusion protein encoded by saidDNA sequence; and (c) isolating the secreted trimeric fusion proteinfrom a host cell.

In a preferred embodiment, the non-collagen polypeptide to be trimerizedis the soluble TNF-RII (p75) (SEQ ID NOS. 9-12 and SEQ ID NOS. 19-20).In another preferred embodiment, the non-collagen polypeptide to betrimerized is soluble CD-4, the co-receptor of HIV (SEQ ID NOS. 13-16).In yet another preferred embodiment, the non-collagen polypeptide to betrimerized is a placental secreted alkaline phosphatase (SEQ ID NOS.5-8).

In one aspect of the invention, a method for producing a secretedtrimeric fusion protein is disclosed, comprising the following: (a)introducing into a eukaryotic host cell a first DNA construct comprisinga promoter which drives the transcription of an open reading frameconsisting of a signal peptide sequence which is linked in-frame to anon-collagen polypeptide to be trimerized, which in turn is joinedin-frame to the C-terminal portion of collagen capable ofself-trimerization, selected from pro.alpha.1(I), pro.alpha.2(I),pro.alpha.1(II), pro.alpha.1(III), pro.alpha.1(V), pro.alpha.2(V),pro.alpha.1(XI), pro.alpha.2(XI) and pro.alpha.3(XI); (b) introducinginto a eukaryotic host cell a second DNA construct comprising a promoterwhich drives the transcription of an open reading frame consisting of asecond signal peptide sequence which is linked in-frame to a secondnon-collagen polypeptide to be trimerized, which in turn is joinedin-frame to the second C-terminal portion of collagen capable ofself-trimerization, selected from pro.alpha.1(I), pro.alpha.2(I),pro.alpha.1(II), pro.alpha.1(III), pro.alpha.1(V), pro.alpha.2(V),pro.alpha.1(XI), pro.alpha.2(XI) and pro.alpha.3(XI); (c) growing thehost cell in an appropriate growth medium under physiological conditionsto allow the secretion of a trimerized fusion protein encoded by saidfirst and second DNA sequences; and (d) isolating the secreted trimericfusion protein from the host cell.

In one aspect of the invention, a method for producing a secretedtrimeric fusion protein is disclosed, comprising the following: (a)introducing into a eukaryotic host cell a first DNA construct comprisinga promoter which drives the transcription of an open reading frameconsisting of a signal peptide sequence which is linked in-frame to anon-collagen polypeptide to be trimerized, which in turn is joinedin-frame to the C-terminal portion of collagen capable ofself-trimerization, selected from pro.alpha.1(I), pro.alpha.2(I),pro.alpha.1(II), pro.alpha.1(III), pro.alpha. 1 (V), pro.alpha.2(V),pro.alpha.1(XI), pro.alpha.2(XI) and pro.alpha.3(XI); (b) introducinginto a eukaryotic host cell a second DNA construct comprising a promoterwhich drives the transcription of an open reading frame consisting of asecond signal peptide sequence which is linked in-frame to a secondnon-collagen polypeptide to be trimerized, which in turn is joinedin-frame to a second C-terminal portion of collagen capable ofself-trimerization, selected from pro.alpha.1(I), pro.alpha.2(I),pro.alpha.1(II), pro.alpha.1(III), pro.alpha.1(V), pro.alpha.2(V),pro.alpha.1((XI), pro.alpha.2(XI) and pro.alpha.3(XI); (c) introducinginto a eukaryotic host cell a third DNA construct comprising a promoterwhich drives the transcription of an open reading frame consisting of athird signal peptide sequence which is linked in-frame to a thirdnon-collagen polypeptide to be trimerized, which in turn is joinedin-frame to a third C-terminal portion of collagen capable ofself-trimerization, selected from pro.alpha.1(I), pro.alpha.2(I),pro.alpha.1(II), pro.alpha.1(III), pro.alpha.1(V), pro.alpha.2(V),pro.alpha.1(XI), pro.alpha.2(XI) and pro.alpha.3(XI); (d) growing thehost cell in an appropriate growth medium under physiological conditionsto allow the secretion of a trimerized fusion protein encoded by saidfirst and second DNA sequences; and (e) isolating the secreted trimericfusion protein from the host cell.

The following are the advantages of this invention: (1) collagen is themost abundant protein secreted in the body of a mammal, constitutingnearly 25% of the total proteins in the body; (2) the major forms ofcollagen naturally occur as trimeric helixes, with their globularC-propeptides being responsible for the initiating of trimerization; (3)the trimeric C-propeptide of collagen proteolytically released from themature collagen is found naturally at sub microgram/mL level in theblood of mammals and is not known to be toxic to the body; (4) thelinear triple helical region of collagen can be included as a linkerwith predicted 2.9 Å spacing per residue, or excluded as part of thefusion protein so the distance between a protein to be trimerized andthe C-propeptide of collagen can be precisely adjusted to achieve anoptimal biological activity; (5) the recognition site of BMP1 whichcleaves the C-propeptide off the pro-collagen can be mutated or deletedto prevent the disruption of a trimeric fusion protein; (6) theC-propeptide domain provides a universal affinity tag, which can be usedfor purification of any secreted fusion proteins created by thisinvention.

In contrast to the Fc Tag technology (Sledziewski et al., 1992 and1998), with which secreted dimeric fusion proteins can be created, thistimely invention disclosed herein enables the creation and secretion ofsoluble trimeric fusion proteins for the first time. Given the fact thata homotrimer has 3-fold symmetry, whereas a homodimer has only 2-foldsymmetry, the two distinct structural forms theoretically can never beperfectly overlaid (FIGS. 1A-1D). As such, neither the homodimericsoluble TNF-R-Fc (e.g. Enbrel®), nor the soluble CD4-Fc fusion proteins,could have had an optimal interface for binding to their correspondinghomotrimeric ligands, TNF-α and HIV gp120, respectively. In contrast,homotrimeric soluble TNF receptors and CD4 created by the currentinvention are trivalent and structurally have the potential to perfectlydock to the corresponding homotrimeric ligands. Thus, these trimericsoluble receptor analogs can be much more effective in neutralizing thebiological activities of their trimeric ligands. With this timelyinvention, more effective yet less expensive drugs, such as trimericsoluble TNF-R and CD4 described in the preferred embodiments, can bereadily and rationally designed to combat debilitating diseases such asarthritis and AIDS. Trimeric soluble gp120 can also be created with thisinvention, which could better mimic the native trimeric gp120 coatprotein complex found on HIV viruses, and used as a more effectivevaccine compared to non-trimeric gp120 antigens previously used. Alsochimeric antibodies in trimeric form can be created with the currentinvention, which could endow greatly increased avidity of an antibody inneutralizing its antigen.

BRIEF DESCRIPTION OF DRAWINGS AND SEQUENCE LISTINGS

FIG. 1A, FIG. 1B, FIG. 1C, and FIG. 1D is a schematic representation ofthe method according to the invention compared to prior dimericimmunoglobulin Fc fusion.

FIG. 1A is a side elevation view and FIG. 1B is a top plan view:Structural characteristics of a homodimeric soluble sTNF RII receptor-Fcfusion, such as Amgen's Enbrel®, in either ligand-free or -bound form asindicated.

Domains labeled in green denote soluble TNF-RII. Note that the Fc(labeled in light blue with inter-chain disulfide bonds in red) fusionprotein is dimeric in structure. Given its 2-fold symmetry, the dimericFc fusion protein is bivalent and thus theoretically does not have theoptimal conformation to bind to a homotrimeric ligand, such as TNF-α(labeled in brown), which has a 3-fold symmetry.

FIG. 1C is a side elevation view and FIG. 1D is a top plan view:Structural characteristics of a trimeric soluble sTNF RIIreceptor-C-propeptide fusion. Given its 3-fold symmetry, a sTNFRII-Trimer fusion protein is trivalent in nature, thus can perfectlydock to its trimeric ligand TNF-α: C-propeptide of collagen capable ofself trimerization is labeled in dark blue with inter-chain disulfidebonds labeled in red.

FIG. 2A and FIG. 2B schematic representation of pTRIMER expressionvectors for creating secreted trimeric fusion proteins. Any solublereceptor- or biological active polypeptide-encoding cDNAs can be clonedinto the unique Hind III or Bgl II sites to allow in-frame fusion at theC-termini to the α (I) collagen containing C-propeptide sequence fortrinmerization. FIG. 2A The pTRIMER(T0) construct contains part of theglycine-repeats (GXY)n upstream of the C-propeptide; FIG. 2B: whereasthe pTRIMER(T2) contains only the C-propeptide domain with a mutatedBMP-1 protease recognition site. pTRIMER(T3) has essentially the samestructure as that of pTRIMER(T2).

FIGS. 3A, 3B, 3C and 3D illustrate the expression and secretion ofdisulfide bond-linked trimeric collagen fusion proteins.

FIG. 3A. Western blot analysis of the trimerization of human placentalalkaline phosphatase (AP) when fused to the C-propeptides of α(I) andα(III) collagens. The expression vectors encoding either AP alone orAP-C-propeptide fusions in pTRIMER vectors were transiently transfectedinto HEK293T cells. Forty-eight hours later, the conditioned media (20μL) of each transfected cells as indicated were boiled for 5 minutes inequal volume of 2×SDS sample buffer either with or without reducingagent (mercaptoethanol), separated on a 10% SDS-PAGE and analyzed byWestern blot using a polyclonal antibody to AP (GenHunter Corporation).Note the secreted 67 kDa AP alone does not form intermolecular disulfidebonds, whereas the secreted AP-T0 and AP-T2 fusions both are assembledefficiently into disulfide bond linked trimers (FIG. 3A, left panel).Similar result with efficient protein trimerization was also obtainedwhen AP was fused to the C-propeptide of human α1(III) collagen (AP-T3)with a mutated putative BMP site (FIG. 3A, right panel).

FIG. 3B. Western blot analysis of the trimerization of soluble humanTNF-RII when fused to the C-propeptides of α(I) and α(III) collagens.The expression vectors encoding either the AP-C-propeptide fusion (T2)(as a negative control for antibody specificity), or human solubleTNF-RII-C-propeptide fusions as indicated in pTRIMER vectors weretransiently transfected into HEK293T cells. Conditioned media fromHEK293T and HEK293T expressing AP-T2 were used as negative controls forTNFRII antibody specificity. Forty-eight hours later, the conditionedmedia (20 μL) of each non-transfected and transfected cells as indicatedwere boiled for 5 minutes in equal volume of 2×SDS sample buffer eitherwith or without reducing agent (mercaptoethanol), separated on a 10%SDS-PAGE and analyzed by Western blot using a monoclonal antibody tohuman TNF-RII (clone 226, R & D Systems, Inc.). Note the monoclonalantibody can only recognize the secreted TNF-RII with disulfide bonds.Soluble TNF-RII-T0, TNF-RII-T2 and TNF-RII-T3 fusions are all assembledefficiently into disulfide bond linked trimers.

FIG. 3C. Trimerized sTNFRII is glycosylated. Two μL of sTNFRII-T2 fromserum-free conditioned medium was digested under denaturing conditionwith either endoglycanase F (PNGase F which digests N-linkedoligosaccharides) or PNGase F plus endo-o-glycosidase which recognizesserine/threonine linked (O-linked) oligosaccharides. The sTNFRII-T2 withand without deglycosylation was analyzed by Western blot usingmonoclonal antibody against TRFRII.

FIG. 3D. Purification of trimeric soluble TNFRII receptor. SolubleTNFRII-T2 was purified to homogeneity from serum-free conditioned mediumof 293T cells stably expressing the fusion protein. Two μg of purifiedprotein was analyzed under either reducing (with β-mercaptoethanol) ornon-reducing conditions by a 10% SDS-PAGE and stained with CoomasieBlue. Note, as shown by western blot analysis shown in FIG. 3B, thepurified soluble TNFRII-T2 fusion protein existed essentially indisulfide bond-linked trimeric form under non-reducing condition.

FIG. 4 and FIG. 5. illustrate the bioassays showing the potentneutralizing activity of the trimeric soluble human TNF-RII-C-propeptidefusion protein against human TNF-α mediated apoptosis.

FIG. 4. The TNF-α sensitive WEHI-13VAR cells (ATCC) were resuspended at1 million cells/mL in RPMI medium containing 10% FBS. 100 μL of the cellsuspension was plated into each well in a 96-well microtiter plate.Actinomycin D was added to each well at 500 ng/mL concentration followedby human TNF-α at 500 pg/ml (R & D Systems) in the presence or absenceof trimeric soluble human TNF-RII-T2 as indicated. As a negativecontrol, the trimeric AP-T2 was added in place of TNF-RII-T2. After 16hours of incubation in a tissue culture incubator, the viability ofcells was examined using either an inverted microscope at 20×magnification or cell viability indicator dye, Alamar Blue (BioSource,Inc.) added to 10% (v/v) to each well. The live cells are able to turnthe dye color from blue to pink. Note that the trimeric soluble humanTNF-RII-T2 exhibits a potent neutralizing activity against TNF-α thecells from TNF-α mediated apoptosis.

FIG. 5. Quantitative analysis of the neutralizing activity of trimericsoluble human TNF-RII-T2 against human TNF-α. The experiment was carriedout as in FIG. 4. Two hours after adding the Alamar Blue dye, theculture medium as indicted from each well was analyzed at OD575. Thereadings were normalized against wells with either no TNF-α (100%viability) added or with TNF-a without neutralizing agent (0% viability)added.

FIGS. 6A and 6B. Comparison of Biological activities of trimeric solublehuman TNF-RII-Trimer fusion protein with dimeric soluble humanTNF-RII-Fc fusion protein in neutralizing TNF-α and against collageninduced arthritis in mice

FIG. 6A. Inhibition of TNF-α mediated apoptosis. TNF-α sensitiveWEHI-13VAR cells were cultured in a 96-well microtiter plate in theabsence or presence of either trimeric sTNFRII-T2 or dimeric sTNFRII-Fcto assess their ability to protect the cells from TNF-α mediatedapoptosis. The experimental conditions were carried out as that in FIG.4. The percentage of the TNF blockers in inhibition of TNF-mediatedapoptosis was normalized against wells where either no TNF-α (100%viability), or with TNF-α without neutralizing TNF blockers (0%viability) added.

FIG. 6B. Comparison of trimeric soluble human sTNFRII-T2 fusion proteinand dimeric sTNFRII-Fc (Enbrel®) in inhibiting collagen-inducedarthritis (CIA) in DBA/1 mice as measured by arthritis scores (mean).The results were representative of 3 independent experiments.

DESCRIPTION OF SEQUENCE LISTINGS

SEQ ID NO: 1 (963 bases)

Nucleotide sequence encoding the C-propeptide human collagen α(I) T0construct. The cDNA construct was cloned into the pAPtag2 vector,replacing the AP coding region.

SEQ ID NO:2 (311 aa)

The predicted C-propeptide T0 protein sequence of human Collagen α(I).The stretch of glycine repeats are located at the N-terminus.

SEQ ID NO:3 (771 bases)

Nucleotide sequence encoding the C-propeptide of human collagen α(I) T2construct. The cDNA construct was cloned into pAPtag2 vector, replacingthe AP coding region.

SEQ ID NO:4 (247 aa)

The predicted C-propeptide T2 protein sequence of human Collagen α(I).The mutated BMP-1 protease recognition site was located at theN-terminus.

(SEQ ID NO:5 (2487 bases)

Nucleotide sequence encoding the human placental alkaline (AP) fused tothe T0 C-propeptide of human α(I) collagen (AP-T0).

SEQ ID NO:6 (819 aa)

The predicted protein sequence of the AP-T0 fusion protein.

SEQ ID NO:7 (2294 bases)

Nucleotide sequence encoding the human placental alkaline phosphatase(AP) fused to the T2 C-propeptide human α(I) collagen (AP-T2).

SEQ ID NO:8 (755 aa)

The predicted protein sequence of the AP-T2 Fusion.

SEQ ID NO:9 (1734 bases)

Nucleotide sequence encoding the human soluble TNF-RII fused to the T0C-propeptide of human α(I) collagen (sTNF-RII-T0).

SEQ ID NO:10 (566 aa)

The predicted protein sequence of the human soluble TNF-RII-T0 Fusion.

SEQ ID NO:11 (1542 bases)

Nucleotide sequence encoding the human soluble TNF-RII fused to the T2C-propeptide of human α(I) collagen (sTNF-RII-T2).

SEQ ID NO:12 (502 aa)

The predicted protein sequence of the human soluble TNF-RII-T2 fusionprotein.

SEQ ID NO:13 (2139 bases)

Nucleotide sequence encoding the human soluble CD4 fused to the T2C-propeptide of human α(I) collagen.

SEQ ID NO:14 (699 aa)

The predicted Protein Sequence of the human soluble CD4-T0 Fusion. Theamino acid residues in blue indicate fusion sites between human solubleCD4 and α(I) collagen T2 polypeptide.

SEQ ID NO:15 (1947 bases)

Nucleotide sequence encoding the human soluble CD4 fused to the T2C-propeptide of human α(I) collagen.

SEQ ID NO:16 (635 aa)

The predicted Protein Sequence of the human soluble CD4-T2 Fusion. Theamino acid residues in blue indicate fusion sites between human solubleCD4 and α(I) collagen T2 polypeptide.

SEQ ID NO: 17 (754 bases)

Nucleotide sequence encoding the C-propeptide of human collagen α1(III)T3 construct with mutated BMP-1 recognition site. The cDNA construct wascloned into pAPtag2 vector at BglII-XbaI sites, replacing the AP codingregion. The flanking sequences denote restriction enzyme sites used inconstructing the corresponding pTRIMER-T3 vector.

SEQ ID NO: 18 (246 aa)

The predicted C-propeptide T3 protein sequence of human Collagen α1(111)with mutated BMP-1 recognition site located at the N-terminus.

SEQ ID NO: 19 (1536 bases)

Nucleotide sequence encoding the human soluble TNF-RII fused to theC-propeptide of human collagen α1(III) T3 construct with mutated BMP-1recognition site.

SEQ ID NO: 20 (501 aa)

The predicted protein sequence of the human soluble TNF-RII fused tohuman collagen α1(III) T3 construct with mutated BMP-1 recognition site.

DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms to beused hereinafter.

DNA Construct: A DNA molecule, generally in the form of a plasmid orviral vector, either single- or double-stranded that has been modifiedthrough recombinant DNA technology to contain segments of DNA joined ina manner that as a whole would not otherwise exist in nature. DNAconstructs contain the information necessary to direct the expressionand/or secretion of the encoding protein of interest.

Signal Peptide Sequence: A stretch of amino acid sequence that acts todirect the secretion of a mature polypeptide or protein from a cell.Signal peptides are characterized by a core of hydrophobic amino acidsand are typically found at the amino termini of newly synthesizedproteins to be secreted or anchored on the cell surface. The signalpeptide is often cleaved from the mature protein during secretion. Suchsignal peptides contain processing sites that allow cleavage of thesignal peptides from the mature proteins as it passes through theprotein secretory pathway. A signal peptide sequence when linked to theamino terminus of another protein without a signal peptide can directthe secretion of the fused protein. Most of the secreted proteins, suchas growth factors, peptide hormones, cytokines and membrane proteins,such as cell surface receptors, contain a signal peptide sequence whensynthesized as a nascent protein.

Soluble receptor: The extracellular domain, in part or as a whole, of acell surface receptor, which is capable of binding its ligand.Generally, it does not contain any internal stretch of hydrophobic aminoacid sequence responsible for membrane anchoring.

C-propeptide of collagens: The C-terminal globular, andnon-triple-helical domain of collagens, which is capable ofself-assembly into trimers. In contrast to the triple helical region ofcollagens, the C-propeptide does not contain any glycine repeat sequenceand is normally proteolytically removed from procollagen precursor uponprocollagen secretion before collagen fibril formation.

Glycine repeats: The central linear triple helix forming region ofcollagen which contains hundreds of (Gly-X-Y)n repeats in amino acidsequence. These repeats are also rich in proline at X or/and Ypositions. Upon the removal of N-and C-propeptides, the glycine-repeatscontaining collagen triple helices can assemble into higher order ofinsoluble collagen fibrils, which make up the main component of the cellmatrix.

cDNA: Stands for complementary DNA or DNA sequence complementary tomessenger RNA. In general cDNA sequences do not contain any intron(non-protein coding) sequences.

One of the modern strategies for treating autoimmune diseases involvesthe use of biologic TNF antagonists, such as soluble receptors ortherapeutic antibodies. However, current TNF-α biologic blockers are alldimeric in structure, whereas TNF-α itself is homotrimeric in nature.Here we describe a general methodology for efficient creation oftrimeric soluble receptors. The process involves gene fusion between asoluble receptor with a ligand binding domain and a trimerization tagfrom the C-propeptide domain of pro-collagen (TRIMER tag), which iscapable of self-assembly into a covalently linked trimer. Using both invitro bioassays and an in vivo mouse model for collagen-inducedarthritis (CIA), we show that the homotrimeric soluble TNF receptorproduced with such method is a more potent blocker than dimeric TNFreceptor decoys in inhibiting TNF-α signaling. Thus, TRIMER tag providesa new platform for rational design of the next generation biologic drugsagainst autoimmune diseases.

Prior to this invention, nearly all therapeutic antibodies and solublereceptor-Fc fusion proteins, such as Enbrel®, are dimeric in structure(FIGS. 1A-1D). Although these molecules, compared to their monomericcounterparts, have been shown to bind their target antigens or ligandswith increased avidity, it is predicted that they are still imperfect,due to structural constrains, to bind their targets that have ahomotrimeric structure. Examples of such therapeutically importanttrimeric ligands include TNF family of cytokines and HIV coat proteingp120. Therefore, from a structural point of view, it will be desirableto be also able to generate trimeric soluble receptors or antibodies,which can perfectly dock to their target trimeric ligands or antigens(FIGS. 1A-1D), and thereby completely block the ligand actions. Suchtrimeric soluble receptors or chimeric antibodies are expected to havethe highest affinity to their targets and thus can be used moreeffectively and efficiently to treat diseases such as arthritis andAIDS.

This invention discloses ways for generating such secreted trimericreceptors and biological active proteins by fusing them to theC-propeptides of collagen, which are capable of self-assembly intotrimers. The following are the advantages of this invention: (1)collagen is the most abundant protein secreted in the body of a mammal,constituting nearly 25% of the total protein in the body; (2) the majorforms of collagen naturally occur as trimeric helixes, with theirglobular C-propeptides responsible for the initiating of trimerization,which are subsequently proteolytically cleaved upon triple helixformation; (3) the cleaved soluble trimeric C-propeptide of collagen isfound naturally at sub microgram/mL level in the blood of mammals; (4)the linear triple helical region of collagen can be included as a linkeror excluded as part of the fusion protein so the distance between aprotein to be trimerized and the C-propeptide of collagen can beprecisely adjusted to achieve an optimal biological activity; (5) therecognition site of BMP1 which cleaves the C-propeptide off thepro-collagen can be mutated or deleted to prevent the disruption of atrimeric fusion protein; (6) the C-propeptides domain provides auniversal affinity tag, which can be used for purification of anysecreted fusion proteins created by this invention; (7) unlike the IgG1Fc tag which is known to be have other biological functions such asbinding to its own cell surface receptors, the only known biologicalfunction of the C-propeptide of collagen is its ability to initiatetrimerization of nascent pro-collagen chains and keep the newly madepro-collagen trimer soluble before assembly into insoluble cell matrix.These unique properties of the C-propeptide of collagen would predictthat this unique trimerization tag is unlikely going to be toxic, orimmunogenic, making it an ideal candidate for therapeutic applications.

To demonstrate the feasibility for making secreted trimeric fusionproteins, cDNA sequences encoding the entire C-propeptides of human al(I) collagen containing either 11 glycine-repeats from the triplehelical region (T0 construct, SEQ ID NOS:1-2), or no glycine-repeat withBMP-1 recognition site (-RADD-) mutated to abolish proteolytic cleavageof the TRIMER tag (T2 construct, SEQ ID NO:3-4) were amplified by PCRusing EST clones purchased from the American Type Culture Collection(ATCC). The amplified cDNAs were each cloned as a Bgl II-XbaI fragmentinto the pAPtag2 mammalian expression vector (GenHunter Corporation;Leder et al., 1996 and 1998), replacing the AP coding region (FIGS. 2Aand 2B). The resulting vectors are called pTRIMER, versions T0 and T2,respectively. Using the same approach, the entire C-prodomain of humanα1(III) collagen with a mutated BMP-1 recognition site and without anyglycine-repeats (T3 construct, SEQ ID No: 17-18) was also amplified byPCR and cloned into pAPtag2 mammalian expression vector. The resultingvector is designated pTrimer-T3. These vectors allow convenient in-framefusion of any cDNA template encoding a soluble receptor or biologicallyactive protein at the unique Hind III and Bgl II sites. Such fusionproteins have the collagen trimerization tags located at the C termini,similar to native pro-collagens.

For trimeric AP and soluble TNFRII protein fusion constructs, the entireAP coding region and cDNA encoding the soluble TNFRII (aa 1-256) withoutthe trans-membrane and cytoplasmic domain were amplified by PCR andcloned into either Hind III or Bgl II sites of pTRIMER expressionvectors to allow in-frame fusion with the TRIMER tags of theC-propeptide. All fusion constructs were verified by DNA sequenceanalysis using an ABI 3100 DNA sequencer.

EXAMPLE 1

To demonstrate the feasibility of this invention, a cDNA encoding thehuman secreted placental alkaline phosphatase (AP), including its nativesignal peptide sequence, was cut out as a Hind III-Bgl II fragment fromthe pAPtag4 vector (GenHunter Corporation; Leder et al., 1996 and 1998)and cloned into the corresponding sites of the pTRIMER-T0, pTRIMER-T2and pTRIMER-T3 vectors. The resulting AP-collagen fusion constructs (SEQID NOS:5-8) were expressed in HEK293T cells (GenHunter Corporation)after transfection. The successful secretion of the AP-collagen fusionproteins can be readily determined by AP activity assay using theconditioned media of the transfected cells. The AP activity reachedabout 1 unit/mL (or equivalent to about 1 μg/mL of the fusion protein) 2days following the transfection. To obtain HEK293T cells stablyexpressing the fusion proteins, stable clones were selected followingco-transfection with a puromycine-resistant vector, pBabe-Puro(GenHunter Corporation). Clones expressing AP activity were expanded andsaved for long-term production of the fusion proteins.

To determine if the AP-collagen fusion proteins are assembled intodisulfide bond-linked trimers, conditioned media containing either APalone or AP-T0, AP-T2 and AP-T3 fusions were boiled in SDS samplebuffers containing either without (non-reducing) or withβ-mercaptoethanol (reducing), separated by an SDS PAGE and analyzed byWestern blot using an anti-AP polycloning antibody (GenHunterCorporation). AP alone without fusion exhibited as a 67 kDa band underboth non-reducing and reducing conditions, consistent with the lack ofany inter-molecular disulfide bonds as expected (FIG. 3A). In contrast,AP-T0, AP-T2 and AP-T3 fusion proteins secreted were shown to be threetimes as big (about 300 kDa) under the non-reducing condition as thoseunder the reducing condition (90-100 kDa), indicating that both fusionproteins were assembled completely into homotrimers (FIG. 3A). Thisresult essentially reduces the concept of this invention to practice.

EXAMPLE 2

To provide a proof that new and therapeutically beneficial biologicalfunctions can be endowed to a trimeric fusion protein, we nextconstructed a trimeric human soluble TNF-RII (p75) receptor using acorresponding EST clone purchased from the ATCC. As described in Example1, the N-terminal region of human TNF-RII, including the signal peptideand the entire ligand-binding region, but excluding the trans-membranedomain, was cloned in-frame, as a Bam H I fragment, into the Bgl II siteof pTRIMER-T0, pTRIMER-T2 and pTRIMER-T3 vectors. The resulting fusionconstructs were expressed in either HEK293T or CHO cells followingtransfection. Stable clones were obtained by puromycine co-selection asdescribed in Example 1. Western blot analysis under both non-reducingand reducing conditions was carried out to determine if the resultingsoluble TNF-RII-collagen fusion proteins were indeed expressed, secretedand assembled into trimeric forms. As expected, the monoclonal antibodyagainst human TNF-RII (clone 226 from R & D Systems, Inc.) clearlyrecognized the trimeric soluble TNF-fusion proteins expressed by allthree (T0, T2 and T3) fusion vectors as 220-240 kDa bands, which areabout three times bigger than the corresponding monomeric fusionproteins (FIG. 3B). The TNF-RII antibody failed to detect monomericfusion proteins under reducing conditions, consistent with the propertyspecified by the antibody manufacturer. As a negative control forantibody specificity, neither the HEK293T cell alone, nor the cellsexpressing AP-T2 fusion protein expressed any TNF-RII (FIG. 3B). Todetermine whether trimerized soluble TNFRII was properly glycosylated,sTNFRII-T2 from serum-free conditioned medium was digested withendoglycanase F (PNGase F which digests N-linked oligosaccharides) andPNGase F plus endo-o-glycosidase which recognizes serine/threoninelinked (O-linked) oligosaccharides. The result shown in FIG. 3C clearlyindicated that the recombinant protein was heavily glycosylated withboth N-and O-linked oligosaccharides, which is evident by the morevisible shift in molecular weight of the minor monomeric form ofsTNFRII-T2 that appeared to be preferentially detected by the westernblot (FIG. 3C). To obtain the sTNFRII-TRIMER in purified form forfunctional analysis, sTNFRII-T from serum-free conditioned medium waspurified to homogeneity essentially following a previously describedprocedure for the purification of the C-propeptide of Type I collagen(Bernocco et al.). As expected, the purified sTNFRII-T2 detected byCoomassie blue staining existed mostly in covalently linked trimericform under non-reducing condition (FIG. 3D).

Expression vectors were either transiently or stably transfected intoeither HEK293T (GenHunter Corp.) or CHO cells (ATTC) using FuGENE 6(Roche). The expression vectors encoding either AP-TRIMER orsTNFRII-TRIMER fusion proteins in pTRIMER vectors were transientlytransfected into HEK293T cells. 48 hours later, the conditioned media(20 μL) of each transfected cells as indicated were boiled for 5 min inequal volume of 2×SDS sample buffer either without or with reducingagent (mercaptoethanol), separated on a 10% SDS-PAGE and analyzed byWestern blot. Secretion of trimeric AP was monitored by both AP activityassay using AP assay A (GenHunter Corp.) and Western blot analysis usingpolyclonal antibody against human placenta AP (GenHunter Corp.).Trimeric soluble TNFRII receptors were analyzed by Western blot usingmonoclonal antibody clone 226 (R & D System) under non-reducingcondition. Deglycosylation of asparagine-linked (N-linked) andserine/threonine-linked (O-linked) carbohydrates from sTNFRII-T2 inserum free conditioned medium was completed with endoglycanase F(PNGaseF) and endo-o-glycosidase (Prozyme), respectively, following theprotocol recommended by the manufacturer. For purification of trimericsoluble TNFRII, 293T cells stably expressing the fusion proteins werecultured first in DMEM with 10% BCS and 1% Pennstrep until confluence.The cells were then switched to HyQ PF293 serum-free medium (Hyclone)after washing 5 times with PBS. After 5-7 days, the conditioned mediawas used as a starting material for purification. The trimeric fusionprotein from 200 mL of conditioned medium was purified to homogeneityusing method essentially as described previously for C-propeptide ofcollagen (Chen, Y., et al.). Purified TNFRII-T2 from the lastpurification step (Butyl-Sepharose) was dialyzed in PBS before used forbiological assays.

To determine if the trimeric soluble TNF-RII receptors are potentinhibitors of its trimeric ligand TNF-α, TNF-α bioassay was carried outusing a cytokine sensitive cell line WEHI-13VAR (ATCC) essentially asdescribed previously (Mohler et al., 1993).

TNF-α bioassays were carried out in duplicate for each data point in a96-well plate using a cytokine sensitive cell line WEHI-13VAR (ATCC)essentially as described previously (Mohler et al. and Khabar et al.).Briefly, exponentially growing WEHI-13VAR cells were resuspended at 1million cells/mL in RPMI medium containing 10% FBS (Hyclone). 100 μL ofthe cell suspension was plated into each well in a 96-well microtiterplate. Actinomycin D was added to each well at 500 ng/mL concentrationfollowed by human TNF-α at 250 pg/ml (R & D Systems) in the presence orabsence of either sTNFRII-Fc (R & D systems, or Amgen) and trimericsoluble human sTNF-RII-T2 as indicated. Both purified sTNFRII-T2 andsTNFRII-Fc were serially diluted in PBS with 1 mg/mL of BSA before used.After 16 hours of incubation in a tissue culture incubator, theviability of cells was examined using either an inverted microscope at20X magnification or cell viability indicator dye, Alamar Blue(BioSource) added to 10% (v/v) to each well. The live cells are able toturn the dye color from blue to pink, which can be quantified at OD575.Percentage in inhibition of apoptosis was normalized against 0% and 100%inhibition without and with TNF-α added only, respectively. Each datapoints were measured from experimental duplicates.

The result shown in FIG. 4 and 5 clearly indicated that the trimericsoluble TNF-RII-C-propeptide fusion proteins are extremely potent inneutralizing the TNF-α mediated apoptosis of WEHI-13VAR cells in thepresence of Actinomycin D (500 ng/mL) (Sigma). When human TNF-α (R & DSystems) was used at 0.5 ng/mL, the trimeric soluble TNF-RII-T2 (bothfrom serum-free media or in purified form) had an apparent Ki-50 (50%inhibition) of about 2 ng/mL or 8×10⁻¹² M (assuming the MW of 240 kDa ashomotrimer). This affinity to TNF-α is 4 orders of magnitude higher thanthat of the monomeric TNF-RII and about 5-10 times higher than that ofthe dimeric soluble TNF-RII-Fc fusion, such as Enbrel® (Mohler et al.,1993) in a side by side comparison (FIG. 6A). Similar results inblocking TNF activity were also obtained for sTNF-RII-T0 and sTNF-RII-T3fusion proteins.

This crucial example proves that this invention can create trimericfusion proteins with new biological properties that may have greattherapeutic applications. Such soluble trimeric human TNF receptors mayprove to be much more effective than the current dimeric soluble TNFreceptor (e.g. Enbrel®) on the market in treating autoimmune diseasessuch as RA. The dramatically increased potency of trimeric-TNF receptorscould greatly reduce the amount of TNF blockers to be injected weeklyfor each patient, while improving the treatment and significantlylowering the cost for the patients. The improved potency of trimeric TNFreceptors should also alleviate the current bottleneck in dimeric TNFreceptor production, which currently can only meet the demands intreating about 100,000 patients in the United States.

EXAMPLE 3

To test the biological activity of the trimeric soluble TNFR in vivo, wethen used the mouse model for collagen-induced arthritis (CIA) (Chen,Y., et al.). For mouse CIA model, the standard protocol with twoimmunization regimens was followed (Chen, Y., et al.). Briefly, 6-8weeks old male DBA/1 mice were purchased from Jackson laboratory andimmunized intradermally each at the base of tail with 100 μg of bovineType II collagen (Condrex) in complete Freund's adjuvant (CFA)(Condrex). The mice were boosted after 3 weeks with 100 μg of bovineType II collagen in incomplete Freund's adjuvant (IFA) (Condrex) toinitiate the CIA. At the same time of boost injections, mice wererandomly divided into 4 treatment groups of 5-6 animals. Each groupreceived either 100 μl of vehicle (PBS), 20 μg of purified trimericsTNFRII-T2 in 100 μl PBS, 20 μg of purified trimeric sTNFRII-T2 in 100μl PBS, or 20 μg of sTNFRII-Fc (Enbrel®) (Amgen, Lot Number: P055643) in100 μl PBS, respectively, all via i.p. injections. Mice were monitoreddaily over a 6 weeks period from the initial immunization for signs ofarthritis. Standard scoring system for arthritis index was followed with0=no swelling, 1=paw swelling with single digit, 2=paw swelling withmultiple digits and 3=severe paw swelling and joint rigidity. Each limbwas graded, giving a maximum possible score of 12 per mice. Statisticalanalysis of daily average arthritis scores was conducted using F teststatistics based on Wilks' Lambda, and their p-values for multivariateprocedures, as recommended by LaTour and Miniard (LaTour and Miniard,1983).

In a side-by-side comparison with the dimeric sTNFRII-Fc fusion protein(Enbrel®) from Amgen®, the trimeric sTNFRII-T2 fusion protein exhibitedsignificant more potent effect, with over 50% inhibition of thearthritis manifestation at any given day post treatment based on meanarthritis scores (P<0.0435), than Enbrel® which gave little protectionas compared with the negative Saline control (FIG. 6B). The minimumeffect observed for Enbrel® could be due to the lower dose (20 μg/mouse) used here than that of previously published (50 μg/mouse) (Wooleyet al.). The lower dose was chosen to best demonstrate the superiority,if any, of the trimeric sTNFRII fusion proteins. If one considers the invitro potency for both the dimeric and trimeric sTNFRII (in the range ofng/ml) in blocking TNF-α, and that each mouse contains no more than 2-3ml of blood, 20 μg/mouse would translate to microgram/mL level of eithertype of TNF blockers if they are completely absorbed into thecirculation. This is about 3 orders of magnitude higher concentrationthan their Kis. Obviously, incomplete absorption into the circulationand rapid turnover (degradation) could significantly affect the finaleffective serum concentration of these fusion proteins. Compared topreviously published CIA studies conducted with either dimeric solubleTNFRII-Fc fusion protein (Wooley et al.) or anti-TNF antibodies(Williams et al.), the trimeric soluble TNFRII also exhibitedsignificantly better effect in suppressing disease severity even whenused at a lower dose.

Taken together, we have demonstrated that with the TRIMER tags frompro-collagens, any soluble receptors or secreted proteins can beefficiently trimerized and expressed as secreted proteins. Such trimericfusion proteins are trivalent in structure with a 3-fold symmetry andthus may have superior biological properties than that of eithernaturally occurring or existing biologic proteins. Such soluble trimerichuman TNF receptors may prove to be more effective than the currentdimeric soluble TNF receptor (e.g. Enbrel®) or therapeutic antibodies onthe market in treating autoimmune diseases such as RA. The increasedpotency of trimeric-TNF receptors has the potential to significantlyreduce the amount of TNF blockers to be injected weekly for eachpatient, while improving the treatment and significantly lowering thecost for the patients. Obviously, such clinical benefits will alsodepend on if a trimeric TNFR soluble receptor has superiorpharmacokinetics properties, such as high stability and low toxicity inthe human body. Future clinical trials should provide such pivotalinformation.

The advantages of using C-propeptide of collagen as a trimerization tagare: (1) collagen is the most abundant protein secreted in the body of amammal, consisting of 25% of total proteins, and the trimericC-propeptide of pro-collagen proteolytically released from the maturecollagen is found naturally in the blood of mammals and is not known tobe toxic to the body; (2) the recognition site of BMP1 which cleaves theC-propeptide off the pro-collagen can be mutated or deleted to preventthe disruption of a trimeric fusion protein; (3) the C-propeptide domainprovides a universal affinity tag, which can be used for purification ofany secreted fusion proteins created with the method; (4) unlike the Fcreceptor, there is no known receptor for the C-propeptides that couldlead to off-target or undesired side effects. Given the fact that ahomotrimer is trivalent and has a 3-fold symmetry, whereas a homodimeris bivalent and has only a 2-fold symmetry, the two distinct structuralforms theoretically can never be perfectly overlaid (FIGS. 1A-1D). Assuch, neither the homodimeric soluble TNFR-Fc (e.g. Enbrel®), nor TNF-αantibodies could have had an optimal interface for binding to theircorresponding homotrimeric ligands, TNF-α. In contrast, homotrimericsoluble TNF receptors created by the TRIMER tag method described herehave the potential structurally to perfectly dock to the correspondinghomotrimeric ligands. Thus, these trimeric soluble receptor analogscould be more effective in neutralizing the biological activities oftheir trimeric ligands, as has been demonstrated here for TNF-α.Conceivably, chimeric antibodies in trimeric form can also be createdwith the TRIMER tag method, which may significantly increased theavidity in neutralizing their trimeric antigens.

It should be pointed out that, unlike Fc tag which binds to protein Atightly, purification of trimerized proteins fused to C-propeptide ofcollagen may be more difficult than Fc fusion proteins. Although it isgenerally believed that TNF family of ligands are mostly homotrimeric insolution or based on x-ray crystallography, biological functional assaysand structural analysis of recombinant TNF expressed in bacteriasuggested that both trimeric and dimeric forms of the ligand couldco-exist (Schoenfeld et al.).

EXAMPLE 4

The HIV virus, the cause of AIDS, infects and destructs primarily aspecial lineage of T lymphocytes in our body. These so called CD4+ Tcells express a cell surface protein dubbed CD4, which is the receptorof HIV. HIV recognizes the CD4+ cells with its viral coat protein gp120that binds to CD4. Notably, the gp120 exists as a giant homotrimericcomplex on the viral surface, whereas the CD4 is monomeric on the cellsurface. The current model for HIV infection is that of a completedocking of HIV to CD4+ T cells, when all three subunits of gp120 trimersare each bound to CD4 is required for viral RNA entry into the cells.Obviously, one of the straightforward strategies for stopping HIVinfection is to use soluble CD4 to blind the virus. Indeed, suchapproach using both monomeric soluble CD4 and CD4-Fc fusions has beenshown quite effective in curbing HIV infections of laboratory isolates(Clapham et al., 1989; Daar et al., 1990). Unfortunately, these solubleCD4 were less effective in stopping the infection of HIV viral strainsfound in AIDS patients (Daar et al., 1990), possibly due to the aminoacid sequence variations of the gp120, which lowers the affinity tomonomeric and dimeric soluble CD4s.

To significantly increase the affinity of a soluble CD4 to any gp120variants on HIV viruses, ideally a soluble CD4 should be in trimericform so it can perfectly dock to its trimeric ligand, gp120 homotrimers.One of the major challenges for combating AIDS has been the highmutational rate of the viral genome, which leads to drug resistance.Therefore any drugs that directly target viral genes, such as HIVreverse transcriptase (e.g. AZT) and protease, are likely renderedineffective as a result of viral mutations. In contrast, no matter howmuch it mutates, a HIV virus has to bind to a cellular CD4 receptor toinitiate the infection. Thus, a high affinity soluble CD4 trimer shouldbe immune to viral mutations because viral mutations in gp120 genes willrender the virus unable to bind not only to a trimeric soluble CD4, butalso CD4 on the cells.

To create such trimeric soluble CD4 HIV receptor analogs, a cDNAencoding the entire human soluble CD4, including its native signalpeptide sequence, but excluding the transmembrane and the shortcytoplasmic domains, was amplified using an EST clone purchased from theATCC. The resulting cDNA was then cloned as a Hind III-Bgl II fragmentinto the corresponding sites of the pTRIMER-T0 and pTRIMER-T2 expressionvectors. The resulting soluble CD4-collagen fusion constructs (SEQ IDNOS:13-16) were expressed in HEK293T cells (GenHunter Corporation) aftertransfection. To obtain HEK293T cells stably expressing the fusionproteins, stable clones were selected following co-transfection with apuromycine-resistant vector, pBabe-Puro (GenHunter Corporation). Clonesexpressing the fusion proteins were expanded and saved for long-termproduction of the fusion proteins.

To determine if the soluble human CD4-collagen fusion proteins areassembled into disulfide bond-linked trimers, conditioned mediacontaining soluble CD4-T0 and CD4-T2 fusions were boiled in SDS samplebuffers containing either without (non-reducing) or withβ-mercaptoethanol (reducing), separated by a SDS PAGE and analyzed byWestern blot using an monoclonal antibody to human CD4 (R & D Systems).Both soluble CD4-T0 and CD4-T2 fusion proteins secreted were shown to bethree times as big (about 300 kDa) under the non-reducing condition asthose under the reducing condition (90-100 kDa), indicating they wereassembled essentially completely into homotrimers (data not shown). Nowthese trimeric soluble CD4 can be readily tested for gp120 binding andanti-HIV infection.

REFERENCES U.S. Patent Documents

-   U.S. Pat. No. 5,155,027, Issued October 1992, Sledziewski et al.-   U.S. Pat. No. 5,605,690, Issued February 1997, Jacobs et al.-   U.S. Pat. No. 5,843,725, Issued December 1998, Sledziewski et al.-   U.S. Pat. No. 6,171,827, Issued January 2001, Bulleid et al.-   U.S. Pat. No. 6,277,600, Issued August 2001, Tomita et al.-   U.S. Pat. No. 5,554,499, Issued September 1996, Leder et al.-   U.S. Pat. No. 5,801,000, Issued September 1998, Leder et al.-   U.S. Pat. No. 6,897,039, Issued May 2005, Graversen et al.

Other References

-   Daar et al. Proc. Natl. Acad. Sci. 87:6574-6578 (1990)-   Alvares et al. Biochemistry 38:5401-5411 (1999)-   Kwong et al. Nature 393:648-659 (1998).-   Clapham et al. British Medical Bulletin 58: 43-59 (2001).-   Clapham et al. Nature 337:368-370 (1989).-   Locksley, R. M., Killeen, N. & Lenardo, M. J. The TNF and TNF    receptor superfamilies: integrating mammalian biology. Cell 104,    487-501 (2001).-   Feldmann, M. & Maini, R. N. Anti-TNF alpha therapy of rheumatoid    arthritis: what have we learned? Annu Rev Immunol. 19, 163-196    (2001).-   Idriss, H. T. & Naismith, J. H. TNF alpha and the TNF receptor    superfamily: structure-function relationship(s). Microsc Res Tech.    50, 184-195 (2000).-   Mohler, K. M. et al. Soluble tumor necrosis factor (TNF) receptors    are effective therapeutic agents in lethal endotoxemia and function    simultaneously as both TNF carriers and TNF antagonists. J Immunol.    151, 1548-1561 (1993).-   Feldmann, M. Development of anti-TNF therapy for rheumatoid    arthritis. Nat Rev Immunol. 2, 364-371 (2002).-   Yang, X., Farzan, M., Wyatt, R. & Sodroski, J. Characterization of    stable, soluble trimers containing complete ectodomains of human    immunodeficiency virus type 1 envelope glycoproteins. J. Virol. 74,    5716-5725 (2000).-   Frank, S. et al. Stabilization of short collagen-like triple helices    by protein engineering. J. Mol. Biol. 308, 1081-1089 (2001).-   Chen, B. et al. A chimeric protein of simian immunodeficiency virus    envelope glycoprotein gp140 and Escherichia coli aspartate    transcarbamoylase. J. Virol. 78, 4508-4516 (2004).-   Stryer, L. in “Connective-Tissue Proteins”, Biochemistry, 3^(rd)    edition, pp261-281 (1989). W. H. Freeman and Company, New York.-   Prockop, D. J., Sieron, A. L. & Li, S. W. Procollagen N-proteinase    and procollagen C-proteinase. Two unusual metalloproteinases that    are essential for procollagen processing probably have important    roles in development and cell signaling. Matrix Biol. 16, 399-408    (1998).-   Li, S. W., Sieron, A. L., Fertala, A., Hojima, Y., Arnold, W. V. &    Prockop, D. J. The C-proteinase that processes procollagens to    fibrillar collagens is identical to the protein previously    identified as bone morphogenic protein-1. Proc Natl Acad Sci U S A.    93, 5127-5130 (1996).-   Melkko, J., Niemi, S., Risteli, L. & Risteli, J. Radioimmunoassay of    the carboxyterninal propeptide of human type I procollagen. Clinical    Chemistry 36, 1328-1332 (1990). Sorva, A. et al. Familial high serum    concentrations of the carboxyl-terminal propeptide of type I    procollagen. Clin Chem. 40, 1591-1593 (1994).-   Bemocco, S. et al. Biophysical characterization of the C-propeptide    trimer from human procollagen III reveals a tri-lobed structure. J    Biol Chem. 276, 48930-48936 (2001).-   Khabar, K. S., Siddiqui, S. & Armstrong, J. A. WEHI-13VAR: a stable    and sensitive variant of WEHI 164 clone 13 fibrosarcoma for tumor    necrosis factor bioassay. Immunol Lett. 46, 107-110 (1995).-   Chen, Y., Rosloniec, E., Price, J., Boothby, M. & Chen, J.    Constitutive expression of BCL-X(L) in the T lineage attenuates    collagen-induced arthritis in Bcl-X(L) transgenic mice. Arthritis    Rheum. 46, 514-521 (2002).-   Wooley, P. H., Dutcher, J., Widmer, M. B. & Gillis, S. Influence of    a recombinant human soluble tumor necrosis factor receptor FC fusion    protein on type II collagen-induced arthritis in mice. J Immunol.    151, 6602-6607 (1993).-   Williams, R. O., Feldmann, M., Maini, R. N. Anti-tumor necrosis    factor ameliorates joint disease in murine collagen-induced    arthritis. Proc Natl Acad Sci U S A. 89, 9784-9788 (1992).-   Schoenfeld, H. J. et al. Efficient purification of recombinant human    tumor necrosis factor beta from Escherichia coli yields biologically    active protein with a trimeric structure that binds to both tumor    necrosis factor receptors. J Biol Chem. 266, 3863-3869 (1991).-   LaTour, S. & Miniard, P. W. The Misuse of Repeated Measures Analysis    in Marketing Research, J. Marketing Res. 20, 45-57 (1983).-   Yang, X., Farzan, M., Wyatt, R. & Sodroski, J. Characterization of    stable, soluble trimers containing complete ectodomains of human    immunodeficiency virus type 1 envelope glycoproteins. J. Virol. 74,    5716-5725 (2000).-   Frank, S. et al. Stabilization of short collagen-like triple helices    by protein engineering. J. Mol. Biol. 308, 1081-1089 (2001).-   Holtet T. L. et al. Tetranectin, a trimeric plasminogen-binding    C-type lectin. Protein Science 6, 1511-1515 (1997).

1. An isolated nucleic acid comprising consecutive nucleotides having anucleotide sequence encoding a fused protein subunit comprising a signalpeptide joined by in-frame fusion to a non-collagenous polypeptidecomprising a ligand binding domain, which in turn is joined by in-framefusion to a C-terminal portion of collagen which is capable ofself-trimerizing said fused protein subunit to form a disulfidebond-linked trimeric fusion protein containing three ligand bindingdomains, wherein said trimeric fusion protein has a greater bindingaffinity to a ligand than a monomeric ligand binding domain.
 2. Thenucleic acid of claim 1, wherein the C-terminal portion of collagenencoded by the nucleic acid is selected from the group consisting ofpro.alpha.1(I), pro.alpha 2(I), pro.alpha.1(II), pro.alpha.1(III),pro.alpha.1(V), pro.alpha.2(V), pro.alpha.1(XI), pro.alpha.2(XI) andpro.alpha.3(XI).
 3. The nucleic acid of claim 1, wherein the signalpeptide and the non-collagenous polypeptide are selected from twodifferent secreted proteins.
 4. The nucleic acid of claim 1, wherein thesignal peptide and the non-collagenous polypeptide are both from anidentical native secreted protein.
 5. The nucleic acid of claim 1,wherein the nucleic acid encodes the C-terminal portion of collagenhaving only a C-propeptide without any linked glycine-repeat triplehelical regions of collagen.
 6. The nucleic acid of claim 1, wherein thenucleic acid encodes the C-terminal portion of collagen having aglycine-repeat triple helical region of collagen linked to aC-propeptide.
 7. The nucleic acid of claim 6, wherein the nucleic acidencodes the C-terminal portion of collagen having an amino acid sequenceshown as SEQ ID NO:2.
 8. The nucleic acid of claim 1, wherein thenucleic acid encodes the C-terminal portion of collagen having a mutatedor deleted BMP-1 protease recognition sequence.
 9. The nucleic acid ofclaim 8, wherein the C-terminal portion of collagen encoded by thenucleic acid is selected from the group consisting of SEQ ID NO:4 andSEQ ID NO: 18.